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Protease Inhibitors in Drug Discovery

2017-04-282018-01-222017-12-21
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The 2018 agenda is currently being formed.

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BELOW IS THE AGENDA FROM 2017.

2018 Agenda
Day 1 - Wednesday, February 8th, 2017
7:00
Continental Breakfast & Registration
7:45
Welcome & Opening Remarks by Dr. Satish Medicetty, President, GTCbio
10:15
Morning Networking Break
 
Marie-Pierre Bousquet-Dubouch
Assistant Professor
University of Toulouse
About Speaker: Education: 1994: Engineer in biochemistry from Institut National des Sciences Appliquées de Toulouse (INSAT). 1994: Master degree in Biotechnology - Microbiology, Institut National des Sciences Appliquées de Toulouse (INSAT). 1997 : Ph.D. in Bio... Read Full Bio 
 
 
Marie-Pierre Bousquet-Dubouch
Assistant Professor
University of Toulouse
 
About Speaker:

Education:

1994: Engineer in biochemistry from Institut National des Sciences Appliquées de Toulouse (INSAT).

1994: Master degree in Biotechnology - Microbiology, Institut National des Sciences Appliquées de Toulouse (INSAT).

1997 : Ph.D. in Biotechnology, Institut National des Sciences Appliquées de Toulouse (INSAT).

Experience:

1992-1994: Industrial trainee at ZENECA BioProducts (Billingham – England) – 18 months

1994-1997: Ph.D. thesis, Laboratory of Prof. P. MONSAN, Laboratoire d’Ingénierie des Systèmes Biologiques et procédés (LISBP): Enzymatic synthesis of alpha-glucosides derivatives for cosmetic applications. Biocatalysis

1997-1999: Postdoctoral and research engineer positions, Centre Régional d’Innovation et de Transfert de Technologies (CRITT, Toulouse). Industrial research contract with ULICE (LIMAGRAIN group).

1999-2002: Assistant Professor of Biochemistry, Laboratory headed by Prof. M.D. Legoy, Laboratoire de Génie Protéique et Cellulaire (LGPC), University of La Rochelle: Fundamentals of gas/solid biocatalysis and application for enantioselective synthesis of chiral synthons.

Since 2002: Associate Professor of Biochemistry at University of Toulouse 3, Senior research scientist at Institut de Pharmacologie et de Biologie Structurale, CNRS, Toulouse. Research expertise in Proteomics and mass spectrometry of biomolecules, structure-activity relationship of human proteasome complexes, study of protein complexes dynamics by quantitative proteomics, protein posttranslational modifications, targeted proteomics.

 
Abstract: Proteasome is a key actor of the ubiquitin-proteasome system. It is formed by the dynamic association of several multimeric sub-complexes, a 20S co...Read More 

Proteasome is a key actor of the ubiquitin-proteasome system. It is formed by the dynamic association of several multimeric sub-complexes, a 20S core particle (20S CP), with one or two regulatory particles (RPs) of identical or different protein composition. The 20S CP can itself be found in the eukaryotic cell as four different sub-types, which differ in their catalytic subunits composition. Proteasomes can also recruit other so-called Proteasome Interacting Proteins (PIPs) which tightly regulate its function. Characterizing the heterogeneity of proteasomes particles would help to better understand the functional relevance of proteasome diversity.

We developed an integrated proteomic workflow based on proteasome immunopurification, label-free quantification using high resolution MS (1), protein correlation profiling, and statistical methods, to resolve the various complexes the different 20S CPs might be involved in and to unravel new proteasome interacting proteins.

From a wide set of human cell lines, previously unreported preferential associations within proteasome sub-complexes could be highlighted. We indeed show that the interactions between the different 20S CPs, RPs, and PIPs do not occur randomly (2). Some of these preferential interactions were validated using complementary approaches. One of these involves PA28?, an ubiquitin-independent 20S CP activator, and a yet uncharacterized protein FAM192A. Using a novel proteomic workflow we identified the specific set of substrates degraded through the 20S-PA28?-FAM192A proteasome sub-complex.

Benefits:

  • Presentation of an integrated proteomic workflow providing a valuable tool to characterize proteasomes sub-populations composition, dynamics, and specific substrates targets.
  • New players in proteasome biology could be unraveled. They can represent valuable therapeutic interest for more targeted therapies compared to classical proteasome inhibitors.
  • Modern proteomic strategies represent complementary alternatives to other structural and cellular biology approaches to characterize the heterogeneity and structure/function relationships of complex molecular systems.
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11:35
Atypical Ubiquitin Chains
 
Eric Strieter
Assistant Professor
University of Massachusetts Amherst
About Speaker: Eric Strieter is an Assistant Professor in the Departments of Chemistry and Biochemistry and Molecular Biology at the University of Massachusetts-Amherst. Eric received his B.S. degree from the University of Wisconsin-Madison. Eric matriculated at MI... Read Full Bio 
 
 
Eric Strieter
Assistant Professor
University of Massachusetts Amherst
 
About Speaker:

Eric Strieter is an Assistant Professor in the Departments of Chemistry and Biochemistry and Molecular Biology at the University of Massachusetts-Amherst. Eric received his B.S. degree from the University of Wisconsin-Madison. Eric matriculated at MIT for graduate studies in 2000 and received his Ph.D. in 2005. His thesis work focused on investigating the mechanistic details of palladium- and copper-catalyzed carbon-nitrogen bond forming reactions. He then was an American Cancer Society postdoctoral fellow at Harvard Medical School with Prof. Chris Walsh where he studied natural product biosynthesis. In 2009, he began his independent career as an Assistant Professor at the University of Wisconsin-Madison. In August 2016 he moved his lab to UMass-Amherst where he continues to investigate the chemistry and biology of protein ubiquitylation.

 
Abstract: The vast functional range of ubiquitin has largely been ascribed to a diverse array of ubiquitin chains. How distinct chain types govern the dynami...Read More 

The vast functional range of ubiquitin has largely been ascribed to a diverse array of ubiquitin chains. How distinct chain types govern the dynamics of different biochemical pathways is poorly understood. Using a library of designer ubiquitin chains we discovered deubiquitinases that are exquisitely selective for certain chain architectures. In this talk we will describe these findings along with their biological implications, specifically in proteasomal degradation.

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12:00
Lunch Provided by GTCbio
Advances in Protease Targeted Therapies
Antoine Dufour, University of British Columbia
1:15
Multi-Omics Analysis of Proteases and Their Inhibitors During Macrophage Polarization
 
Antoine Dufour
Postdoctoral Fellow, Chris Overall Lab
University of British Columbia
About Speaker: Antoine Dufour, Ph.D., is a post-doctoral fellow funded by the Canadian Institutes of Health Research (CIHR) in the laboratory of Chris Overall, the pioneer of degradomics, at the University of British Columbia. He received his doctoral degree from S... Read Full Bio 
 
 
Antoine Dufour
Postdoctoral Fellow, Chris Overall Lab
University of British Columbia
 
About Speaker:

Antoine Dufour, Ph.D., is a post-doctoral fellow funded by the Canadian Institutes of Health Research (CIHR) in the laboratory of Chris Overall, the pioneer of degradomics, at the University of British Columbia. He received his doctoral degree from Stony Brook University in chemical biology working on the development of exosite inhibitors (patent STONYB-16639) for the inhibition of matrix metalloproteinases during cancer cell migration, invasion, angiogenesis and tumor metastasis. In 2014, Dr. Dufour was the chair of the Gordon Research Seminar “Proteolytic Enzymes & Their Inhibitors” in Il Ciocco, Italy. He currently studies the role of various proteases in immune regulation, viral response, cancer and autoimmune/inflammatory diseases using degradomics and proteomics.

 
Abstract: The mononuclear phagocyte system is a highly dynamic and complex system that can be unified based upon progenitor cells but disjointed based upon t...Read More 

The mononuclear phagocyte system is a highly dynamic and complex system that can be unified based upon progenitor cells but disjointed based upon the stimuli they are responding to. Classically activated (M1) macrophages (TH1) are induced alone or in combination by interferon (IFN) ? and lipopolysaccharide, whereas alternatively activated M2 macrophages (TH2) differentiate by several activators including interleukin (IL) 4, IL-13, IL-10, glucocorticoids, immune complexes, and transforming growth factor ? (TGF-?). Proteases acting in concerted networks to amplify signals and regulate biology are hypothesized molecular switches and effectors involved in macrophage differentiation and functions. THP-1 and U-937 monocytic human cell lines along with murine RAW264.7 and in vivo peritoneal macrophages were used to study the differentiation between macrophage populations: M0 (untreated), M1 (IFN-?) and M2 (IL-4). Multi-omics analysis using protease-specific microarrays (CLIP-CHIP), quantitative proteomics (SILAC) and terminomics (TAILS) was performed to characterize differentiation in the context of proteolytic processing. Alignment with CLIP-CHIP arrays narrowed protease markers across the three populations to CATL1, LAP3 and MMP9 for M1 macrophages and azurocidin for M2. Our biological validations demonstrated that both murine and human macrophages require serine protease activity for migration, however, only the M1 macrophages required cysteine protease activity for migration. M1 macrophages are highly dependent on proteases to regulate phagocytosis whereas M0 and M2 macrophages were not inhibited by leupeptin, E64, marimastast, pepstatin A or AEBSF. In conclusion, we developed an integrated multidimensional approach to characterize proteolytic activity using omics technologies revealed protease groups involved in macrophage differentiation and biological functions.

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1:40
Identification of Mouse Cathepsin K structural Elements That Regulate the Potency of Odanacatib
 
Dieter Brömme
Professor, Canada Research Chair, Oral Biological and Medical Sciences
University of British Columbia
About Speaker: Dieter Brömme is a professor and Canada Research Chair in Proteases and Disease at the Department of Oral Biological and Medical Sciences of the University of British Columbia in Vancouver/Canada. He received his Ph.D. from the Martin-Luther-Univers... Read Full Bio 
 
 
Dieter Brömme
Professor, Canada Research Chair, Oral Biological and Medical Sciences
University of British Columbia
 
About Speaker:

Dieter Brömme is a professor and Canada Research Chair in Proteases and Disease at the Department of Oral Biological and Medical Sciences of the University of British Columbia in Vancouver/Canada. He received his Ph.D. from the Martin-Luther-University (MLU) in Halle(Saale)/GDR in 1983. Since his post-graduate studies at the MLU he carried on his interest in lysosomal cysteine proteases and their role in human pathology throughout his career which included positions at the Biotechnology Research Institute in Montreal, at Khepri/Arris Pharmaceuticals in South San Francisco, and at the Mount Sinai School of Medicine in New York before joining the University of British Columbia.

 
Abstract: Cathepsin K (CatK) is the predominant mammalian bone-degrading protease and thus an ideal target for anti-osteoporotic drug development. Rodent mod...Read More 

Cathepsin K (CatK) is the predominant mammalian bone-degrading protease and thus an ideal target for anti-osteoporotic drug development. Rodent models of osteoporosis are preferred due to their close reflection of the human disease and their ease of handling, genetic manipulation, and economic affordability. However, large differences in the potency of CatK inhibitors for the mouse/rat versus the human protease orthologues have made it impossible to use rodent models. This is even more of a problem considering that the most advanced CatK inhibitors including odanacatib and balicatib failed in human clinical trials due to side effects and rodent models are not available to investigate the mechanism of these failures. Here, we elucidated the structural elements of the potency differences between mouse and human CatK using odanacatib (ODN). We determined and compared the structures of inhibitor-free mouse CatK (mCatK), human CatK (hCatK) and ODN bound to hCatK. Two structural differences were identified and investigated by mutational analysis. Humanizing subsite 2 in mCatK led to a 5-fold improvement of ODN binding whereas the replacement of Tyr61 in mCatK with Asp resulted in an hCatK with comparable ODN potency. Combining both sites further improved the inhibition of the mCatK variant. Similar results were obtained for balicatib. These findings will allow the generation of transgenic CatK mice that will facilitate the evaluation of CatK inhibitor adverse effects and to explore routes to avoid them.

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2:05
The Proteasome and Fibroblast Activation Protein in Liver Fibrosis
 
Mark Gorrell
Molecular Hepatology Laboratory Head, Centenary Institute of Cancer Medicine & Cell Biology
University of Sydney
About Speaker: Associate Professor Mark Gorrell of the Centenary Institute, University of Sydney, trained in cell biology, virology, immunology and protein biochemistry at Australian National University, University of Melbourne and Johns Hopkins University. His res... Read Full Bio 
 
 
Mark Gorrell
Molecular Hepatology Laboratory Head, Centenary Institute of Cancer Medicine & Cell Biology
University of Sydney
 
About Speaker:

Associate Professor Mark Gorrell of the Centenary Institute, University of Sydney, trained in cell biology, virology, immunology and protein biochemistry at Australian National University, University of Melbourne and Johns Hopkins University. His research is focussed upon liver cancer prevention and treatment, chronic liver disease pathogenesis, diabetes, protein and protease biochemistry and cell biology related to the proteases DPP4, DPP9 and fibroblast activation protein (FAP). He has authored over 130 publications attracting >5,000 citations and H index 38.

 
Abstract: Severe liver fibrosis (scarring) can be caused by prolonged liver damage of any aetiology, including haemochromatosis, autoimmune disease, viral in...Read More 

Severe liver fibrosis (scarring) can be caused by prolonged liver damage of any aetiology, including haemochromatosis, autoimmune disease, viral infection, excessive ethanol consumption or overnutrition. Severe fibrosis is reversible, but if persistent can result in organ failure or cancer. Liver fibrosis is mainly driven by innate responses to tissue damage, primarily hepatocyte and biliary cell apoptosis, that activates fibroblasts and macrophages. Current therapeutic targets include drivers of fibroblast proliferation, such as TGF-beta, PDGF, CTGF and certain kinases. Activated fibroblasts are the major sources of the abnormal extracellular matrix (ECM) that forms the scar. A recent approach showing promise targets lysyl oxidase-like 2, which crosslinks collagen. Fibroblast activation protein (FAP) is made at high levels by activated hepatic myofibroblasts and is a collagenase involved in regulating cell-ECM interactions. We have observed that the FAPgko mouse is healthy, has a normal response to influenza infection and has normal populations of splenic leukocytes; including splenic dendritic cells, B cells, neutrophils, and CD4+ and CD8+ T cells. However, in the fibrotic liver FAPgko mice have fewer B cells and CD4+ T cells and less fibrosis. In contrast, in a lung fibrosis model FAP is anti-fibrotic.

Proteasome: The proteasome inhibitor bortezomib is anti-apoptotic for hepatocytes and anti-proliferative for liver fibroblasts. However, the known adverse side effects of bortezomib preclude it from use as an anti-fibrotic agent in humans. We used a FAP-activated bortezomib-like prodrug to deliver proteasome inhibition to FAP-positive liver fibroblasts in a fibrosis model. Livers from FAP-activated prodrug treated mice contained less FAP, collagen-I, TNF-?, TGF-?, IL6 and MT1-MMP, consistent with the expected depletion of myofibroblasts. Hepatocytes appeared histologically normal in prodrug treated livers but had extensive ballooning in controls with fibrosis.

Conclusion: Depleting FAP-expressing cells and delivering intrahepatic proteasome inhibition in mice depresses the intrahepatic expression of pro-fibrotic molecules. Thus, FAP has potential for directing therapies such as proteasome inhibition towards activated fibroblasts and thereby reducing liver fibrosis.

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2:30
Advances in Protease Targeted Therapies
 
Tobias Kromann-Hansen
PhD
University of California San Diego
About Speaker: Tobias Kromann-Tofting, PhD., is currently a Postdoc at the Department of Chemistry and Biochemistry at University of California San Diego. Tobias Kromann-Tofting received his PhD in Molecular Biology from Aarhus University, Denmark. Tobias Kromann-T... Read Full Bio 
 
 
Tobias Kromann-Hansen
PhD
University of California San Diego
 
About Speaker:

Tobias Kromann-Tofting, PhD., is currently a Postdoc at the Department of Chemistry and Biochemistry at University of California San Diego. Tobias Kromann-Tofting received his PhD in Molecular Biology from Aarhus University, Denmark. Tobias Kromann-Tofting’ research is concentrated on the development of inhibitors against trypsin-like serine proteases. Working with the protease urokinase-type plasminogen activator (uPA), which is involved in progression of diseases such as arthritis and cancer, Tobias Kromann-Tofting has developed new principles for intervention with the catalytic activity of uPA by studying a new type of protease inhibitors namely singe domain antibody fragments from Camelids (Alpacas, Camels, Dromedaries and Llamas).

 
Abstract: Trypsin-like serine proteases play pivotal roles in human physiology regulating processes such as blood coagulation, fibrinolysis, digestion and co...Read More 

Trypsin-like serine proteases play pivotal roles in human physiology regulating processes such as blood coagulation, fibrinolysis, digestion and complement activation. A trypsin-like serine protease of particular interest is urokinase-type plasminogen activation (uPA), which under normal physiological conditions is involved in tissue remodeling processes such as wound healing. Under pathophysiological conditions, however, the catalytic activity of uPA is involved in progression of diseases such as arthritis, cancer and atherosclerosis. Thus, targeting the catalytic activity of uPA represent an attractive strategy for pharmacological intervention with its pathophysiological activities. We have developed new strategies for selective inhibition of uPA using Camelid-derived single domain antibody fragments (nanobodies). During this talk I will present biochemical and biophysical data to show how nanobodies can be used as orthosteric (active site binding) as well as allosteric (exosite binding) inhibitors of uPA. Moreover, I will show how two different nanobodies can be fused into one multivalent inhibitor for effective targeting of multiple functional activities of uPA.

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2:55
Afternoon Networking Break
Advances in Protease Targeted Therapies (Cont’d)
Daniel Bachovchin, Memorial Sloan Kettering Cancer Center
3:25
DPP8/9 Inhibition Induces Pro-caspase-1-dependent Pyroptosis
 
Daniel Bachovchin
Assistant Member, Chemical Biology
Memorial Sloan Kettering Cancer Center
About Speaker: Daniel Bachovchin, Ph.D., is an Assistant Member in the Chemical Biology Program at Memorial Sloan Kettering Cancer Center. The Bachovchin lab uses chemical biology approaches to study the roles that proteases play in cancer and immune system signali... Read Full Bio 
 
 
Daniel Bachovchin
Assistant Member, Chemical Biology
Memorial Sloan Kettering Cancer Center
 
About Speaker:

Daniel Bachovchin, Ph.D., is an Assistant Member in the Chemical Biology Program at Memorial Sloan Kettering Cancer Center. The Bachovchin lab uses chemical biology approaches to study the roles that proteases play in cancer and immune system signaling. Dr. Bachovchin performed graduate research at The Scripps Research Institute with Ben Cravatt and postdoctoral research at The Broad Institute with Todd Golub.

 
Abstract: Val-boroPro (talabostat, PT-100), a nonselective inhibitor of post-proline cleaving serine proteases, stimulates mammalian immune systems through a...Read More 

Val-boroPro (talabostat, PT-100), a nonselective inhibitor of post-proline cleaving serine proteases, stimulates mammalian immune systems through an unknown mechanism of action. Despite this lack of mechanistic understanding, Val-boroPro has attracted significant interest as a potential anticancer agent, reaching Phase III trials in humans. Here I will discuss our recent finding that Val-boroPro stimulates the immune system by triggering a proinflammatory form of cell death in monocytes and macrophages known as pyroptosis. We have demonstrated that the inhibition of two serine proteases, DPP8 and DPP9, activates the proprotein form of caspase-1 independent of the inflammasome adaptor ASC. Activated pro-caspase-1 does not efficiently process itself or IL-1?, but does cleave and activate gasdermin D to induce pyroptosis. Mice lacking caspase-1 do not show immune stimulation after treatment with Val-boroPro. Our data identifies the first small molecule that induces pyroptosis and reveals a new checkpoint that controls the activation of the innate immune system.

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3:50
MALT1 and Therapeutic Modulation of Linear Ubiquitin in Autoimmunity
 
Christopher M. Overall
Canada Research Chair in Protease Proteomics and Systems Biology
Centre for Blood Research, University of British Columbia
About Speaker: Dr. Overall is a Professor and Canada Research Chair in Protease Proteomics and Systems Biology, U.B.C. Vancouver. With 23 Nature Review, Science, and Nature/Cell/Science-sister journal papers (h-index 67), he is a pioneer of degradomics, a term he c... Read Full Bio 
 
 
Christopher M. Overall
Canada Research Chair in Protease Proteomics and Systems Biology
Centre for Blood Research, University of British Columbia
 
About Speaker:

Dr. Overall is a Professor and Canada Research Chair in Protease Proteomics and Systems Biology, U.B.C. Vancouver. With 23 Nature Review, Science, and Nature/Cell/Science-sister journal papers (h-index 67), he is a pioneer of degradomics, a term he coined. He completed his Ph.D. at the University of Toronto; and post-doctoral work with Dr. Michael Smith, Nobel Laureate. In 1997/1998 was a Visiting Senior Scientist at British Biotech, Oxford and in 2004/2008 a Visiting Senior Scientist at Novartis, Basel, and is now an Honorary Professor, Albert-Ludwigs Universität Freiburg. Dr. Overall was 2002 CIHR Scientist of the Year, the UBC Killam Senior Researcher Award 2005, and was Chair of the 2003 MMP and the 2010 Protease Gordon Research Conferences. He was recognized by the IPS with the 2011 Lifetime Achievement Award; by the Matrix Biology Society of Australia and New Zealand with the 2012 Barry Preston Award; and in 2014 by the Tony Pawson Canadian National Proteomics Network Award for Outstanding Contribution and Leadership to the Canadian Proteomics Community. He is also an elected member of HUPO Executive Committee, the Chromosome Centric Human Proteome Project (C-HPP) Executive Committee, and is an Associate Editor of the Journal of Proteomics Research.

4:15
Deciphering Proteases in Regulated Cell Death
 
Guy Salvesen
Professor, Tumor Microenvironment and Cancer Immunology Program
NCI-Designated Cancer Center
About Speaker: ... Read Full Bio 
 
 
Guy Salvesen
Professor, Tumor Microenvironment and Cancer Immunology Program
NCI-Designated Cancer Center
 
About Speaker:
 
Abstract: The objective of this talk is to highlight the proteases required for regulated cell death mechanisms in animals – apoptosis, regulated necr...Read More 

The objective of this talk is to highlight the proteases required for regulated cell death mechanisms in animals – apoptosis, regulated necrosis, necroptosis, pyroptosis, and NETosis. Each of these death mechanisms has at least one proteolytic component that plays a critical role in controlling the pathway, and sometimes they combine in networks to regulate cell death/survival decision nodes.

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4:40
Kvido Strisovsky
Principal Investigator
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic
About Speaker: Kvido Strisovsky, PhD, is a group leader at the Institute of Organic Chemistry and Biochemistry (IOCB) in Prague, Czech Republic. He received education at Charles University, Prague, and at Cambridge University and MRC Laboratory of Molecular Biology... Read Full Bio 
 
 
Kvido Strisovsky
Principal Investigator
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic
 
About Speaker:

Kvido Strisovsky, PhD, is a group leader at the Institute of Organic Chemistry and Biochemistry (IOCB) in Prague, Czech Republic. He received education at Charles University, Prague, and at Cambridge University and MRC Laboratory of Molecular Biology, United Kingdom. Dr. Strisovsky studies the mechanism and roles of intramembrane proteolysis in biological regulation. Most notably, he discovered the principles of substrate recognition by rhomboid proteases, his group was the first to solve a structure of an intramembrane protease with a fragment of its substrate and is a pioneer in the development of rhomboid protease inhibitors. Dr. Strisovsky is a member of the EMBO Young Investigator Programme.

5:05
Networking Reception & Poster Session
5:05
Targeting the Proprotein Convertase Furin for Novel Potential Broad Spectrum Therapeutics
 
Maurizio Pellecchia
Professor of Biomedical Sciences Daniel Hays Endowed Chair in Cancer Research
UC Riverside School of Medicine
About Speaker: Dr. Pellecchia is a chemical biologist with a strong background in pharmaceutical chemistry, biophysics and translational medicine. He trained at the University of Naples (Italy) where he obtained a MS in Organic Chemistry and a Ph.D. in Pharmaceutic... Read Full Bio 
 
 
Maurizio Pellecchia
Professor of Biomedical Sciences Daniel Hays Endowed Chair in Cancer Research
UC Riverside School of Medicine
 
About Speaker:

Dr. Pellecchia is a chemical biologist with a strong background in pharmaceutical chemistry, biophysics and translational medicine. He trained at the University of Naples (Italy) where he obtained a MS in Organic Chemistry and a Ph.D. in Pharmaceutical Sciences, at the ETH-Zurich (working with 2002 Nobel Laureate Prof. Dr. Kurt Wüthrich) and the University of Michigan. Prior to his recruitment in 2002 at the Burnham Institute for Medical Research as Associate Professor, he spent a few years in the pharmaceutical industry. He has served on the faculty of the now Sanford Burnham Prebys medical Discovery Institute for 14 years where he also served as the Associate Director for Translational Research for the Institute’s NCI designated Cancer Center.

Since 2015 he is a Professor of Biomedical Sciences at the University of California at Riverside, School of Medicine and I hold the Daniel Hays endowed Chair in Cancer Research. In addition is the Director of the Center for Molecular and Translational Medicine at UCR. His research is at the forefront of academic drug discovery andchemical biology initiatives. His goals are to support target identification and validation studies in oncology, neurodegenerative, and infectious diseases. The laboratory focuses primarily on the development of innovative pharmacological agents and subsequently apply such agents in target validation studies using cellular and animal models, both internally and via collaborations. Central to these activities are the developing and the application of novel methods and strategies to drug discovery and translational medicine.

 
Abstract: Furin is a proprotein convertase (PC) involved in numerous pathogenic processes including viral propagation and bacter...Read More 

Furin is a proprotein convertase (PC) involved in numerous pathogenic processes including viral propagation and bacterial toxin activation. In addition to normal cellular functions, PCs such as furin process to maturity membrane fusion proteins and pro-toxins of a variety of bacteria and viruses, including anthrax and botulinum toxins, influenza A H5N1 strain, and potentially other viruses including flaviviruses, Marburg, Dengue and Zika viruses. Therefore, inhibition of furin would in principle provide significant host protection against multiple unrelated furin-dependent pathogens. Based on furin’s substrate preferences and the recently derived X-ray structure of furin in complex with a covalent peptide-based inhibitor, dec-RVKR-CMK, we designed and synthesized selective compounds with nanomolar affinity and tested the activity of the agents in cellular assays against Anthrax toxin, Dengue, Ebola and other viruses. Preliminary pharmacokinetic and toxicity studies are also reported as well in vivo efficacy studies with a toxemia mouse model of Anthrax toxin. Our studies shed more light on the potential application of furin inhibitors as therapeutics potentially against various furin-dependent pathogens.

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Day - 2 Thursday, February 9th, 2017
7:30
Continental Breakfast
Protease Inhibitors in Translational Research
Mark Gorrell, University of Sydney
8:00
Immunoproteasome Selective Inhibitor Promotes Long-Term Cardiac Allograft Acceptance
 
Gang Lin
Associate Professor of Research in Microbiology and Immunology
Cornell University
About Speaker: My lab focuses on developing species-selective or isoform-selective proteasome inhibitors for diseases, such as Tuberculosis, malaria, autoimmune and inflammatory disorders.... Read Full Bio 
 
 
Gang Lin
Associate Professor of Research in Microbiology and Immunology
Cornell University
 
About Speaker:

My lab focuses on developing species-selective or isoform-selective proteasome inhibitors for diseases, such as Tuberculosis, malaria, autoimmune and inflammatory disorders.

8:25
Usp9x: Molecular Target in Cancer
 
Luke Peterson
Assistant Research Scientist
University of Michigan
About Speaker: PhD from the University of Manchester UK, Postdoc The Scripps Research Institute La Jolla, CA University of Michigan since 2007... Read Full Bio 
 
 
Luke Peterson
Assistant Research Scientist
University of Michigan
 
About Speaker: PhD from the University of Manchester UK, Postdoc The Scripps Research Institute La Jolla, CA University of Michigan since 2007
 
Abstract: The ubiquitin cycle is a crucial protein regulatory system controlling many aspects of cell signaling, growth and death. Conjugation of ubiquitin t...Read More 

The ubiquitin cycle is a crucial protein regulatory system controlling many aspects of cell signaling, growth and death. Conjugation of ubiquitin to proteins is reversible via a sub-family of ~100 deubiquitinases (DUBs). Growing evidence suggest that the DUB Usp9x plays a role in cancer biology, as it positively regulates the stability of oncogenes such as Mcl-1, β-catenin and Erg. Targeting Usp9x activity is thus an attractive avenue to counteract activities of oncogenic factors. We previously showed in multiple myeloma (MM) and blast crisis chronic myeloid leukemia (CML) cells, that Usp9x regulates their growth and survival. To characterize targets of Usp9x we used an unbiased proteomic approach to identify/quantify ubiquitin protein remnants (UbiScan) post the loss of Usp9x activity in melanoma cells. We identified that Usp9x regulates proteins involved in transcription, Ras signaling, and negative regulators of signaling, such as phosphatases. Specifically, Usp9x controls the stability of the oncogene Ets-1. Importantly, the Ets-1/Usp9x axis directly controls the transcription of the N-Ras protein. In addition, Usp9x and Ets-1 levels are coincidently elevated in melanoma. In particular, Ets-1 induction by BRAF or MEK kinase inhibition resulted in increased NRAS expression that was blocked by inactivation of Usp9x. In vivo therapeutic combination of Usp9x and a MEK inhibitor fully suppressed melanoma growth. Lastly, we identified that in breast cancer cells the same targetable Usp9x/Ets-1 axis is functional. Therefore, Usp9x might be an essential target in in various Ets-1 driven cancers.

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8:50
Ectosteric Inhibitors of Cathepsin K Inhibits Bone Resorption in Ovariectomized Mice
 
Preety Panwar
Research Associate, OBMS
University of British Columbia
About Speaker: Preety Panwar, PhD., is a research associate in Dieter Bromme’s group at University of British Columbia. She has background in liposomes mediated drug delivery. Currently, she is investigating the mechanism of cathepsin K mediated extracellular mat... Read Full Bio 
 
 
Preety Panwar
Research Associate, OBMS
University of British Columbia
 
About Speaker:

Preety Panwar, PhD., is a research associate in Dieter Bromme’s group at University of British Columbia. She has background in liposomes mediated drug delivery. Currently, she is investigating the mechanism of cathepsin K mediated extracellular matrix degradation, and identification of specific anti-collagenase inhibitors of cathepsin K and their in vitro and vivo evaluation.

 
Abstract: The potent Cathepsin K (CatK) inhibitor, tanshinone IIA Sulfonic Sodium (T06), was tested for its in vitro and in vivo antiresorp...Read More 

The potent Cathepsin K (CatK) inhibitor, tanshinone IIA Sulfonic Sodium (T06), was tested for its in vitro and in vivo antiresorptive activity. In contrast to all other known CatK inhibitors, T06, binds in a so-called ectosteric site causing the selective inhibition of collagen degradation with an IC50 value of 2.1 ± 0.3 μM (protease:T06 molar ratio of 1:5) but does not suppress gelatinolysis at molar CatK to T06 concentrations of 1:2,500. T06 suppressed bone resorption by human osteoclasts with an IC50 value of ~210 nM, and that of mouse osteoclasts with an IC50 value of ~225 nM without affecting the metabolic activity of osteoclasts. The antiresorptive activity of T06 was fully reversible. Moreover, osteoclastogenesis was not affected by T06 at the 4-fold antiresorptive IC50 concentration. In in vivo studies using 12-weeks old ovariectomized (OVX) mice, vehicle and T06 (40 mg/kg) was orally administered for three months to sham and OVX operated mice. μCT analysis of femur and vertebra of T06-treated OVX mice revealed a significant reduction in bone mineral density loss and an improvement of trabecular bone parameters when compared to control animals. Serum CTx was also reduced but T06 did not decrease the numbers of OCs, had no estrogenic effect on the uterus, and no effect on fibroblast mediated TGF-β degradation, and cognitive functions. This study indicates that an ectosteric inhibitor like T06 is an attractive alternative to active site inhibitors which all have failed in clinical trial.

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9:15
Covalent Versus Non-Covalent Protease Inhibitors in Drug Discovery – A Proteomics Perspective
 
Benedikt Kessler
Professor of Biochemistry and Life Science Mass Spectrometry
Oxford University
About Speaker: ... Read Full Bio 
 
 
Benedikt Kessler
Professor of Biochemistry and Life Science Mass Spectrometry
Oxford University
 
About Speaker:
 
Abstract: Covalent or irreversible protease inhibitors have been developed and successfully applied in the past for a range of c...Read More 

Covalent or irreversible protease inhibitors have been developed and successfully applied in the past for a range of clinical applications. However, predominantly because of pharmacokinetic, pharmacodynamic and safety concerns, there has been a reluctance to consider covalent molecules in target-specific drug discovery projects. Covalent or irreversible inhibitors react to the enzyme and exert very slow off-rates that are exceeding the time of protein re-synthesis rates. Recent deep proteomics data sets including large-scale protein turnover studies using stable isotope labelling of amino acids in cell culture (SILAC) now provide insights in to decay rates of entire protein families. Interestingly, proteases of the different hydrolase sub-families have a half-live within a range of a few hours to more than ten days with many of them having a turnover of more than 24-48 hours. These rates are often slower than what is observed for drug metabolic turnover, and therefore would suggest that irreversible inhibitors for a long-lived protease may have favorable pharmacokinetic properties in a clinical setting.

Benefits:
– Discussing the use of reversible versus irreversible protease inhibitors in drug discovery
– Deep proteomics metabolic labeling data suggests slow turnover rates for many proteases
– Slow protease turnover rates might be an advantage for the clinical use of irreversible inhibitors

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9:40
Morning Networking Break
Protease Inhibitors in Translational Research (Cont’d)
Rama K. Mallampalli, University of Pittsburgh
10:10
Small Molecules Targeting F-box Proteins to Modulate Inflammation
 
Rama K. Mallampalli
UPMC Endowed Professor and Division Chief, Pulmonary, Allergy, and Critical Care Medicine
University of Pittsburgh
About Speaker: Dr. Mallampalli is a UPMC Endowed Professor and Division Chief of Pulmonary Allergy, and Critical Care Medicine at the University of Pittsburgh School of Medicine. He work is internationally recognized in the pathogenesis of sepsis and pneumonia as i... Read Full Bio 
 
 
Rama K. Mallampalli
UPMC Endowed Professor and Division Chief, Pulmonary, Allergy, and Critical Care Medicine
University of Pittsburgh
 
About Speaker:

Dr. Mallampalli is a UPMC Endowed Professor and Division Chief of Pulmonary Allergy, and Critical Care Medicine at the University of Pittsburgh School of Medicine. He work is internationally recognized in the pathogenesis of sepsis and pneumonia as it relates to acute lung injury (ALI). His research has discovered a unique model for the molecular behavior of ubiquitin E3 ligase subunits belonging to the Skp-Cullin1-F box (SCF) family that control inflammation. Dr. Mallampalli’s laboratory designed, synthesized, and tested a new genus of ubiquitin E3 ligase (F box) inhibitors that modulate proteolysis thereby inhibiting inflammation in preclinical models of ALI and multi-organ failure.

 
Abstract: Cytokine-driven inflammation is a fundamental pathophysiological process for diverse infectious and immune-related dis...Read More 

Cytokine-driven inflammation is a fundamental pathophysiological process for diverse infectious and immune-related disorders. We discovered that a ubiquitin E3 ligase F box subunit, termed Fbxo3, potently stimulates cytokine secretion from human inflammatory cells by triggering the degradation of the TNF receptor associated factor (TRAF) inhibitory protein, Fbxl2. By using in silico modeling we designed and tested a unique small molecule targeting the carboxyl-terminal domain of Fbxo3. The small molecule Fbxo3 inhibitor effectively reduced TRAF protein levels, potently inhibited cytokine release from human blood mononuclear cells, and lessened the severity of inflammation in several preclinical models. Studies assessing pharmacokinetics demonstrate properties consistent with a druggable compound in animal studies. Thus, pharmacological targeting of Fbxo3 appears to be a new strategy for disorders characterized by a heightened host inflammatory response.

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10:35
Pharmacological and Toxicological Properties of the Potent Oral Gamma-Secretase Modulator BPN-15606
 
Steven Wagner
Associate Professor, Neuroscience
University of California San Diego
About Speaker: Dr. Steven Wagner PhD is an Associate Professor in the Department of Neurosciences at UCSD. He has spent over 25 years in the biopharmaceutical industry, and more recently in academia studying translational neuroscience of age-related neurodegenerati... Read Full Bio 
 
 
Steven Wagner
Associate Professor, Neuroscience
University of California San Diego
 
About Speaker:

Dr. Steven Wagner PhD is an Associate Professor in the Department of Neurosciences at UCSD. He has spent over 25 years in the biopharmaceutical industry, and more recently in academia studying translational neuroscience of age-related neurodegenerative disorders with an emphasis on Alzheimer’s disease (AD). He led the team that discovered the first non-NSAID-like and truly “Notch-sparing” gamma-secretase modulators and introduced the term “gamma-secretase modulators” (GSMs) in 2005 through the discovery of a novel series of diaryl-2-aminothiazole derivatives that are over 5000-fold more potent at lowering A42 levels than the NSAID-like “substrate-targeted” gamma-secretase modulators, e.g., tarenflurbil. His team also, for the first time, purified to homogeneity the gamma-secretase enzyme complex that is ultimately responsible for generating amyloid β(Aβ) plaques, the diagnostic hallmark of AD. Since moving back to academia, into the Department of Neurosciences at UCSD in June of 2009, his laboratory, in addition to designing/discovering another novel and structurally distinct GSM chemotype, was awarded a Blueprint Neurotherapeutics U01 by NIH/NINDS (one of only seven issued in all of Neurology) to optimize and develop GSMs for the treatment and/or prevention of AD. He is also a member of the NIH Drug Discovery SBIR (ETTN-M)ETTN IRG, Division of Neuroscience, Development and Aging Study Section, a member of the NINDS Special Emphasis Panel for the Blueprint Neurotherapeutics Network, as well as a member of the Cure Alzheimer’s Fund (CAF) Research Consortium and the Scientific Advisory Board for the Alzheimer’s Association’s Collaboration for Cure (C4C).

 
Abstract: Inhibitors of γ-secretase have been developed for Alzheimer’s disease (AD) with the aim of decreasing brain levels of all Aβ peptide species (A...Read More 

Inhibitors of γ-secretase have been developed for Alzheimer’s disease (AD) with the aim of decreasing brain levels of all Aβ peptide species (Aβ42, Aβ40, Aβ39, Aβ38 and Aβ37). Based on the fact that the vast majority of the more than 200 FAD-linked genetic mutations appear to cause a 2-fold increase in the ratio of the longer more oligomeric-prone Aβ42 peptide relative to the shorter Aβ40 peptide, and a large body of data pointing specifically to Aβ42 in AD pathogenesis, a therapeutic rationale that modulates γ-secretase activity to reduce only the level of Aβ42 without affecting overall γ-secretase activity may prove most efficacious. We view gamma-secretase modulation as superior to the use of vaccines and passive immunization which require invasive procedures and immune regulation. In addition, because gamma-secretase modulators (GSMs) directly attenuate the level of Aβ42, while increasing levels of shorter Aβ peptides (Aβ38 and Aβ37) they may prove to be easier to test, evaluate and monitor clinically than compounds which inhibit the activities of either β-secretase (BACE inhibitors such as verubecestat are currently in phase 3 clinical trials), γ-secretase or both. To date GSMs have yet to be tested in AD patients due primarily to adverse events encountered during phase 1 safety studies. We have identified a very potent GSM, BPN-15606, with an excellent pharmacological and toxicological profile which we feel may be suitable ultimately for testing in AD.

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11:00
The Study and Promise of Deubiquitinases as Therapeutic Targets
 
Johanna Heideker
PhD
Genentech
About Speaker: Dr. Heideker received her PhD in 2012 from The Scripps Research Institute, La Jolla, CA, studying the functions of a SUMO-targeted ubiquitin ligase in DNA repair and genome maintenance in the laboratory of Michael (Nick) Boddy. From there she joined ... Read Full Bio 
 
 
Johanna Heideker
PhD
Genentech
 
About Speaker:

Dr. Heideker received her PhD in 2012 from The Scripps Research Institute, La Jolla, CA, studying the functions of a SUMO-targeted ubiquitin ligase in DNA repair and genome maintenance in the laboratory of Michael (Nick) Boddy. From there she joined Ingrid E. Wertz' lab at Genentech as a Postdoctoral researcher, where she set out to explore novel roles of deubiquitinases (DUBs) in human disease. During her time at Genentech in collaboration with the Proteomics and Biostatistics departments she build a novel quantitative platform to measure deubiquitinase activity in a semi-native environment that can be used to both identify new cellular modes of DUB regulation as well as aid the development of compounds targeting DUB activity. Her assay is now being used across GNE departments to aid the study of DUBs and support drug discovery efforts targeting the ubiquitin-proteasome system.

 
Abstract: Deubiquitinases (DUBs, ubiquitin proteases) are important regulators of cell physiology and emerging drug targets due to their critical roles in ce...Read More 

Deubiquitinases (DUBs, ubiquitin proteases) are important regulators of cell physiology and emerging drug targets due to their critical roles in cellular biology. However, the exact function or regulation of the majority of the estimated 100 human DUBs remains largely enigmatic and new members of this family continue to be discovered. DUB activity-based probes are engineered ubiquitin variants conjugated to a cysteine-reactive chemical group. We envisioned that DUB activity-based probes coupled with mass spectrometry may provide a highly sensitive and unbiased means to discover novel DUB function, dissect DUB regulatory mechanisms, and aid DUB-based therapy development. To compare DUB activity across theoretically unlimited sample numbers we here combine DUB activity-based probes, quantitative mass spectrometry techniques with internal reaction standards and a novel data analysis and visualization platform. The methodology allows us to quantitate changes in DUB activity across a multitude of different selected samples that are assayed in separate experiments. We provide case studies that illustrate the efficacy of this technology to facilitate the discovery of disease-relevant functions of DUBs and modes of DUB activity regulation, and reveal DUB regulation by ubiquitin-proteasome modulating compounds.

BENEFITS of the talk: insight into DUBs as drug targets, insight into DUB activity regulation, example of interdisciplinary quantitative assay development, insight into mass-spectrometry based quantitation of enzyme activity, insight into compounds modulating DUB activity.

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Round Table Discussions
12:25
Lunch Provided by GTCbio
Joint Session: Translational Assay Development
Roland Wolkowicz, San Diego State University
1:35
Go With the Flow: Development of in Vitro Potency Tests for Stem Cell Therapies
 
Sofie PattjIn
CTO
ImmunXperts SA
About Speaker: Sofie Pattijn (CTO and founder, ImmunXperts) has over 20 years of experience in the field of immunogenicity assessment (vacci nes and biotherapeutics) and in vitro assay development. She has extensive hands-on lab experience and has managed and coach... Read Full Bio 
 
 
Sofie PattjIn
CTO
ImmunXperts SA
 
About Speaker:

Sofie Pattijn (CTO and founder, ImmunXperts) has over 20 years of experience in the field of immunogenicity assessment (vacci nes and biotherapeutics) and in vitro assay development. She has extensive hands-on lab experience and has managed and coached several In Vitro teams over the last decade. From 2008 till 2013 she was Head of the In Vitro Immunogenicity group at AlgoNom ics (Ghent, Belgium) and Lonza Applied Protein Services (Cambridge, UK). Prior to that, she worked at Innogenetics, Belgium for over 15 years.

 
Abstract: Cellular therapies are becoming more and more important for the treatment of cancer, autoimmune disorders, hematologic malignancies and tissue dama...Read More 

Cellular therapies are becoming more and more important for the treatment of cancer, autoimmune disorders, hematologic malignancies and tissue damage. As this field develops, the ability to produce large quantities of biological products with predictable quality and quantifiable potency is of great importance. Potency testing is the quantitative measure of a biological activity which is linked to relevant biological properties of a product. The biological activity measured should be closely related to the product’s intended biological effect and ideally it should be related to the product’s clinical response. Technologies such as flow cytometry contribute to the development and application of complex in vitro assays representing the in vivo biological activity of the cellular therapy. The use of well characterized primary immune cells in combination with flow cytometry and other techniques such as ELISA can result in the availability of a robust and easy to implement screening and release assay. Additionally, these assays are also a valuable tool in elucidating the mechanism underlying for example Stem Cell immunomodulatory functions.

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2:00
3D Cell Screening Assays Using Automated Microinjection
 
Jan de Sonneville
CEO
Life Science Methods B.V.
About Speaker: Born on 14-09-1980 in Amsterdam, the Netherlands. Bachelor in Electrical Engineering, Delft University of Technology (TUDelft), Master in NanoScience (Applied Physics), given as joint program by TUDelft and Leiden University, the Netherlands (2006). ... Read Full Bio 
 
 
Jan de Sonneville
CEO
Life Science Methods B.V.
 
About Speaker:

Born on 14-09-1980 in Amsterdam, the Netherlands. Bachelor in Electrical Engineering, Delft University of Technology (TUDelft), Master in NanoScience (Applied Physics), given as joint program by TUDelft and Leiden University, the Netherlands (2006). PhD on the development of four novel research methods for Cell Biology, thesis title: Reinventing microinjection, new microfluidic methods for cell biology (2011). Founded Life Science Methods BV to sell Automated Microinjection Systems for high throughput screening using cell spheroids and zebrafish embryos (2011).

 
Abstract: Using an automated injection robot, we inject tiny droplets of cells into a hydrogel. With this system, we performed a screen of com...Read More 
  • Using an automated injection robot, we inject tiny droplets of cells into a hydrogel.
  • With this system, we performed a screen of compounds that affect breast cancer cell migration, and demonstrated speed, reproducibility, and compatibility with existing lab equipment and techniques.
  • In another screen, we found a correlation of prostate cancer cell migration patterns with metastasis in a mouse model, as well as a dosage dependent effect of a new kinase inhibitor. This kinase was also shown to be expressed in patient tissue.
  • Cell-cell and cell-matrix interactions have been studied by injecting two different cell types next to each other in the same gel, paving the way towards more complex biological models.
  • Currently, we are exploring interactions between immune cells and cancer cells, and immune cells and cells with a bacterial infection.
  • The principle of injecting droplets of cells into precisely predefined spots in a matrix offers many possible applications that I would love to discuss.
  • We can 3D print any tissue of interest. As such our niche is quite broad, especially compared to other competing 3D tissue systems.
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2:25
Monitoring Cleavage Towards and at the Cell Surface for the Enhancement of Drug discovery
 
Roland Wolkowicz
Professor, Director of the FACS Facility
San Diego State University
About Speaker: Roland Wolkowicz, Ph.D. is a Professor in Biology at San Diego State University in SD, CA. Born in Barcelona, Spain, he pursued undergraduate research in Biology at the University of Tel Aviv, Israel. Obtained his MSc in Microbiology from Tel Aviv Un... Read Full Bio 
 
 
Roland Wolkowicz
Professor, Director of the FACS Facility
San Diego State University
 
About Speaker:

Roland Wolkowicz, Ph.D. is a Professor in Biology at San Diego State University in SD, CA. Born in Barcelona, Spain, he pursued undergraduate research in Biology at the University of Tel Aviv, Israel. Obtained his MSc in Microbiology from Tel Aviv University, and PhD in Molecular Cell Biology from the Weizmann Institute of Science in Rehovot, Israel, where he studied the p53 DNA binding activity. As a postdoctoral fellow at Stanford University, he became acquainted with retroviral technology, peptide libraries and flow cytometry-based biological screenings. As a research associate at Stanford, he studied novel ways to block HIV-1 infection. In 2006, he joined the Department of Biology at San Diego State University, where he also serves as the Director of the FACS Core facility. His laboratory investigates viral-host interactions, focusing mainly on HIV-1 and Flaviviridae members such as HCV, Dengue virus, West Nile virus and Zika. His laboratory studies the effect of infection on host signaling cascades, and develops cell-based assays that monitor proteolytic cleavage for drug discovery.

 
Abstract: Proteolysis is an essential biological process utilized, among many others, for protein activation, degradation of aggregates, and regulation of si...Read More 

Proteolysis is an essential biological process utilized, among many others, for protein activation, degradation of aggregates, and regulation of signaling cascades. Many proteolytic events occur within the vesicles of the secretory pathway, comprising the Endoplasmic Reticulum, Golgi and trans-Golgi Network (ER/Golgi/TGN) that link the nucleus to the cellular membrane. Monitoring cleavage within the classical secretory pathway or at the cell surface in a biologically relevant background can enhance drug discovery efforts against less obvious targets. These targets include the HIV-1 envelope or premature membrane protein (prM) of Dengue virus (DenV), cleaved within the Golgi/TGN by Furin (or similar), or matrix metalloproteinases such as MMP-14, overexpressed in some cellular transformations and metastasis and active at the cell surface or extracellular matrix. We have previously developed an assay that monitors cleavage of the gp120/gp41 HIV-1 envelope boundary, which was then adapted to the DenV prM boundary and used in a pilot screen in search for inhibitors/competitors of prM cleavage. As proteins such as MMP-14 exploit the same secretory pathway for their own activation and for their transport to the surface, we decided to further adapt the assay to specifically monitor cleavage at the cell surface rather than in its route to the surface. The original assay was based on an engineered protein embedded into the ER membrane consisting of a two-tag system flanking the DenV prM boundary substrate. The assay could distinguish between cleaved and non-cleaved events based on classical flow cytometry. An optimized substrate of MMP-14 was then used as a proof-of-principle to monitor cleavage by MMP-14 at the cell surface. Constitutive/inducible MMP-14 overexpression showed cleavage of the substrate. Preliminary mixing experiments of cells expressing the substrate with cell expressing MMP-14 corroborated that cleavage specifically occurred at the cell surface. This novel platform represents a powerful tool for the search of inhibitors/competitors of MMP-14 substrate recognition/cleavage and is easily adaptable to any substrate cleaved at this cellular location. Benefits: – Cell-based assays that monitor cleavage at the cell surface are limited. – Assay monitors cleavage in the cellular compartment where it naturally occurs. – Adaptable to multiplexed format. – Adaptable to 96 and 384 well-plate for HTS in a robust and stable cell line.

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2:50
Afternoon Networking Break
Joint Session: High Throughput and High Content Screening Assays
Michael Mancini, Baylor College of Medicine
3:20
Phenotypic 3D Imaging of Living Biopsies for Cancer Chemoresistance Screening
 
David Nolte
Distinguished Professor, Physics
Purdue University
About Speaker: David D. Nolte is the Edward M Purcell Distinguished Professor of Physics and Astronomy at Purdue University performing research in the fields of optical technologies for molecular diagnostics and cancer therapeutics. He received his baccalaureate fr... Read Full Bio 
 
 
David Nolte
Distinguished Professor, Physics
Purdue University
 
About Speaker:

David D. Nolte is the Edward M Purcell Distinguished Professor of Physics and Astronomy at Purdue University performing research in the fields of optical technologies for molecular diagnostics and cancer therapeutics. He received his baccalaureate from Cornell University in 1981, his PhD from the University of California at Berkeley in 1988, and was a post-doctoral member of AT&T Bell Labs before joining the physics faculty at Purdue. He has been elected Fellow of the Optical Society of America, Fellow of the American Physical Society and Fellow of the AAAS. In 2005 he received the Herbert Newby McCoy Award of Purdue University. He has founded two biotech startup companies in the area of diagnostic screening and high-content analysis.

 
Abstract: Three-dimensional cancer biopsies from patients are imaged using biodynamic imaging based on laser ranging and Doppler frequency spectroscopy. The ...Read More 

Three-dimensional cancer biopsies from patients are imaged using biodynamic imaging based on laser ranging and Doppler frequency spectroscopy. The response of the living biopsy samples to applied therapeutics is captured as shifts in the biodynamic spectra, presenting specific resistant or sensitive phenotypes. This phenotypic screen captures the physiological response of the tissue to the therapy without the need for genetic profiling. Several therapy candidates can be screened simultaneously, providing oncologists with information on best response for individual patients. This aid to therapy selection can help bring personalized cancer care closer to practice.

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3:45
Single Cell Analysis of Steroid Receptor Functions by HCA
 
Michael A. Mancini
Professor
Baylor College of Medicine
About Speaker: ... Read Full Bio 
 
 
Michael A. Mancini
Professor
Baylor College of Medicine
 
About Speaker:
 
Abstract: The main focus of our research has been to identify, characterize and quantify mechanistic steps of steroid nuclear receptor action at the single c...Read More 

The main focus of our research has been to identify, characterize and quantify mechanistic steps of steroid nuclear receptor action at the single cell level by using state-of-the-art microscopy based approaches. To this end, we created stable cell lines and automated image analysis routines that facilitate multi-parametric small molecule screens to identify estrogen and androgen receptor effectors, RNAi libraries for pathway analysis, and also endocrine disruptor compound (EDC) screening. These efforts have led to the creation of biological response fingerprints that identify mechanistic and phenotypic changes in biosensor and native cell lines in response to various treatments. We have also identified novel, disease relevant estrogen receptor coregulators (i.e. UBR5) and receptor-specific EDCs (Bisphenol A analogs). These recent efforts are leading towards an improved understanding of steroid receptor and coregulator action in prostate and breast cancer models.

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4:10
Unfolding the Unfolded Protein Response
 
Chris Wilson
Associate Director
Small Molecule Discovery Center, UCSF
About Speaker: Chris Wilson is Associate Director of screening at the Small Molecule Discovery Center, UCSF Mission Bay,. He was previously a Senior Scientist at Ensemble Therapeutics (Cambridge, MA) and a postdoctoral fellow at Yale University, in the protein engi... Read Full Bio 
 
 
Chris Wilson
Associate Director
Small Molecule Discovery Center, UCSF
 
About Speaker:

Chris Wilson is Associate Director of screening at the Small Molecule Discovery Center, UCSF Mission Bay,. He was previously a Senior Scientist at Ensemble Therapeutics (Cambridge, MA) and a postdoctoral fellow at Yale University, in the protein engineering group of Prof. Lynne Regan.

 
Abstract: The Small Molecule Discovery Center (SMDC) at UCSF couples a broad technology base, with a highly collaborative philosophy, to tackle novel screens...Read More 

The Small Molecule Discovery Center (SMDC) at UCSF couples a broad technology base, with a highly collaborative philosophy, to tackle novel screens against challenging targets and biological pathways. Through transcriptional and translational reporter approaches, three independent branches of the Unfolded Protein Response (UPR) were probed, each serving as mutual counter-screens in hit selection. Subsequent medicinal chemistry and animal studies have revealed the potential of the UPR in modulating disease.

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Joint Session: Stem Cell Based Screening Assays
Michael Mancini, Baylor College of Medicine
4:35
Phenotypic Screening of Human Induced Pluripotent Stem Cell (hiPSC) Derived Neuronal Networks On Multi-Electrode Arrays
 
Anne Bang
Director, Cell Biology
Sanford Burnham Prebys Medical Discovery Institute
About Speaker: Anne Bang joined the Sanford Burnham Prebys in June 2010 as Director of Cell Biology at the Conrad Prebys Center for Chemical Genomics, a state-of-the-art drug discovery center. Her efforts there are directed at developing patient-specific, and human... Read Full Bio 
 
 
Anne Bang
Director, Cell Biology
Sanford Burnham Prebys Medical Discovery Institute
 
About Speaker:

Anne Bang joined the Sanford Burnham Prebys in June 2010 as Director of Cell Biology at the Conrad Prebys Center for Chemical Genomics, a state-of-the-art drug discovery center. Her efforts there are directed at developing patient-specific, and human induced pluripotent stem cell (hiPSC)-based models that reflect higher order cellular functions and disease phenotypes, yet have the throughput and reproducibility required for drug discovery and target identification. Prior to joining SBP she was at ViaCyte Inc. where, as Director of Stem Cell Research, she managed an interdisciplinary group of scientists working to develop hESC as a replenishable source of pancreatic cells for the treatment of diabetes. Dr. Bang has over 20 years of experience in the fields of developmental and stem cell biology, with a focus on neural development. She received a B.S. from Stanford, a Ph.D. in Biology from UCSD, and was a post-doctoral fellow at the Salk Institute.

 
Abstract: Human induced pluripotent stem cells (hiPSC) could aid in the development of clinically useful compounds. They allow interrogation of differentiate...Read More 

Human induced pluripotent stem cells (hiPSC) could aid in the development of clinically useful compounds. They allow interrogation of differentiated features of human cells, circumvent issues of species specificity, and importantly, carry disease-specific traits in complex genetic backgrounds that can impact disease phenotypes. Development of patient specific hiPSC based models to study the cellular and molecular bases of neurological disease offers an opportunity to identify improved treatments, and to better stratify patients according to pathological processes. The biology and genetics underlying neurological disease are complex and will require the examination of multiple cell types from many patient hiPSC lines to identify and validate phenotypes. Development of procedures to interrogate hiPSC derived neural cells in miniaturized higher-throughput formats will be advantageous not only for drug screening, but also for phenotype discovery, allowing testing of multiple lines and variables, such as timing and dose response to therapeutic agents, pathway modulators, and stress inducers. Using hiPSC-derived neurons, we have developed a functional assay on 48 and 96 multi-well multi-electrode array (MEA) plates that recapitulates physiologically relevant synchronized bursting properties sensitive to shifts in the balance of excitation and inhibition. Advantages of this system include throughput, and the ability to non-invasively measure networked neuronal activity over days and months. We will discuss use of this assay for compound screening and modeling of synaptic plasticity and neurological disease.

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5:00
Conference Concludes